Modern structural biology seeks to establish a molecular understanding of biological function by exploring structure-function relationships in biomolecules. The mechanisms for protein folding, protein-protein interactions, ligand binding and catalysis are fundamentally linked to structure. Membrane proteins, in particular, drive key biological processes such as cell-signaling, and ion transport through the cell membrane. Despite the abundance, diversity, and biological importance of membrane proteins, relatively few crystal structures have been solved due to the difficulty in crystallizing the proteins. Modern tools such as cryo-electron-microscopy or x-ray crystallography enable detailed measurements of protein structure on multiple length-scales; however, structures are usually measured under non-biological conditions where key dynamic effects that lead to protein stability and function are not captured. Aside from NMR spectroscopy, most bio-analytical tools in use today are not sensitive to protein structure, heterogeneity, or conformational dynamics. Specific classes of proteins such as fibrous proteins, intrinsically disordered proteins, gels, amyloids, and aggregates, have been particularly difficult to characterize due to a lack of structural techniques available.
Infrared spectroscopy in the amide I region has been used to measure protein structure in solution. The vibrational modes in these regions involve primarily backbone C═O and N—H vibrations that are relatively sensitive to secondary structure and are largely free from the influence of side-chain absorptions. To date, however, most infrared measurements have offered either qualitative information or have required significant efforts to vibrationally isolate individual residues through isotope labeling. Attempts to extract structural information from amide-I and II absorption bands in proteins have relied on complex deconvolution and fitting to analyze largely featureless absorption bands. The results of these measurements are heavily dependent on the specific set of frequencies and fitting functions used, which greatly limits the usefulness of linear infrared spectroscopy as a structural tool.